PROJECT SUMMARY/ABSTRACT Senescent cells (SCs) have emerged as a potential therapeutic target for aging and age-related diseases. Senolytic agents, small molecules that can selectively kill SCs, have the potential to be developed as ?anti- aging? drugs that can extend human healthspan (years of life without disability or other impairment) by preventing or treating age-related diseases. Recently, we reported the discovery of ABT-263 (navitoclax) as a potent and selective senolytic agent through screening of a library of rationally selected compounds. Our studies show that ABT-263 functionally rejuvenates hematopoietic stem cells (HSCs) in both sublethally irradiated (6 Gy) and naturally aged mice by depleting SCs, including senescent HSCs, thus demonstrating the therapeutic potential of senolytic agents. Following the same approach, we have identified a second senolytic agent, piperlongumine (PL), a natural dietary product isolated from a variety of Piper (pepper) species. Unlike ABT-263, which induces SC apoptosis by inhibiting Bcl-2 proteins, the underlying molecular mechanisms of action (MMOA) of PL in inducing SC death are undefined, raising the potential of discovering novel molecular targets for senolytic drug development. Therefore, we hypothesize that PL can serve as a promising lead for the development of novel senolytic agents and can be used to identify molecular targets that are important for SC survival. The objectives of this application are to discover and evaluate PL analogues (?piperlogues?) as potent and selective senolytic agents, identify their molecular targets, and elucidate the MMOA of these novel agents. These objectives will be accomplished by carrying out the following specific aims: 1) design, synthesize and evaluate novel piperlogues to improve the senolytic activity of PL; 2) identify and validate the protein targets of PL and piperlogues in SCs, and elucidate their MMOA; and 3) evaluate the in vivo efficacy of lead piperlogues. Success in these proposed aims will not only produce PL-based anti-aging drug development candidates but also identify and validate novel molecular target(s) to enable future design and target-based discovery of novel senolytic agents and to gain more insights into the biology of cellular senescence and the MMOA of PL and piperlogues.